Process of preparing remedial serum.



' NITED STATES" PATENT: OFFICE.

FRANZ NIEMANN, OF BASLE, SWITZERLAND.

PROCESS OF PREPARING REMEDIAL SERUM.

srEcIF'IcATION forming part of Letters Patent a... 651,951, dated June 19, 1eoo.

Application filed March 20, 1896- Serial No. 534,023. (No specimens.)

To all whom it may concern:

Be it known that I, FRANZ NIEMANN, of Basle, Switzerland, have invented certain new and useful Improvements in the Manufacture of Remedial Serum, of which the following is a specification.

This invention has reference to the manufacture of remedial serum, and more particularly of serum for the cure or retardation of the progress of tuberculosis.

Since the discovery of tuberculin by Koch frequent attempts have been made to prepare a tuberculin-antitoxine for remedial purposes. Among the earlier attempts injections of pure living cultivations of tubercle bacilli were made into horses and other animals and blood drawn from the tuberculous animals after several weeks. The serum separated from this blood was stated to possess a slight specific antitoxic action on tuberculous sub-. jects; but the animal serving for the preparation of the serum almost invariably died before any considerable'quantity of antitoxine was elaborated in its system, and consequen tly experiments in this direction were too few for any definite result to be arrived at. In'place of tubercle bacilli injections were then tried of tuberculin, which is a sterilized glycerine extract of pure cultures of the tubercle bacillus. Boinet and E. Managliano have stated thatit is possible by repeated injections with tuberculin, of which no quantities are given, to obtain a serum from the inoculated animal capable of exerting a remedial effect on tuberculous subjects. It appears, however, from their reports that only a brief temporary delay in the progress of the disease was afforded by the use of this serum, and Managliano himself admits that its inj ection is objectionable in advanced stages of consumption or where the infected area is extensive, while Boinet states that the treatment is of no avail in the advanced disease associated with excavation and is injurious even in tuberculosis of acute stage. The employment of tuberculin alone for injection, therefore, appears as futile as the employment of tubercle bacilli in the manufacture of antitoxic serum of remedial value.

After careful consideration of the probable causes of failure and studying the disadvantages, both practical and theoretical, of the above methods I succeeded after many trials in discovering a procedure different from the foregoing which enables tuberculous remedial serum to be prepared of high antitoxic value. Especially was I led to my conclusions by the consideration that glycerine is a blood poison, and when introduced into the circulation partially dissolves or shrivels up the red corpuscles and causes disturbances and stoppages in the blood-vessels, especially in the neighborhood of the pulmonary or gans, which set up conditions promoting parenchymat-ous change. It therefore appeared to me that only very small quantities of til berculin could be injected without running the risk of developing deleterious by-efiects from glycerine poisoning. For the preparation of remedial serum according to the new method it is preferred to employ one to two year old goats, though other animals-such as horses, asses, mules,

&c.--may be employed should it be found im practicable to manufacture sufiicient quantities of serum from the smaller animals. In

the following directions for effecting immunity the quantities therefore only refer to healthy goats twenty-four kilos in weight, but the proportions of materials used to theanimal weight remain substantially identical ascertained by keeping it under close observation for about four weeks prior to the commencement of the treatment, during which period the weight of the animal should not decrease, but rather increase.

In carrying out this invention the treatment to which I subject the animals in order to procure the desired degree of immunity and the elaboration of a sufficient quantity of tuberculin-antitoxine in their systems to render the preparation of my remedial serum possible should be effected in three distinct stages, which I term the preliminary treatment, the special treatment, and the final treatment, respectively.

Preliminary treatment. -A subcutaneous injection of two cubic centimeters tuberculin is made, preferably, in the neck of the animal by means of a sterilized Pravatz syringe or other suitable instrument. The tuberculin which I employ for this purpose is preftoo erably made in the following manner: A pure cultivationoftubercle bacilli from tubercu; lous human organs is made in gl'ycerine-goatblood serum, and then a sterilized bouillon lated bouillon. is then kept in an incubator for eight weeks at 41 centigrade. If at the end of that period a thick crust of tubercle bacilli ha's'forme'd' upon the surface of the bouillon and microscopical examination 're- Veals the complete purity of the cultivation, the culture is sterilized by heating for two hoursat 100 ccntigrade and then concen trated-to one-tenth of its bulk filtered through aGham-berland filter and the filtrate evapo-j rated to one-half, The dark yellow thick liquidso obtained is the tuberculin employed inthe'preliminary'treatnient. injection of-two cubic centimeters of this tu berculin there'is usually a rise in tempera ture in goats of 1 to 1 centigrade. forty-eight hours a second injection of four cubic centimeters tuberculin is made, and if there'is no subsequent rise in temperature within twenty-four hours. at the end of fortyeight hours after the second injection athlrd injection of four cubic centimeters tuberculin is made. If, on'the other hand, areaction occurs after twenty-four hours, the third injeetion: is unnecessary. The preliminary treatmentiis now complete, andI proceed'(after aninterval ofabout eight to ten days) tothespecial treatment, for whichI employ the precipitated active compound or principle of tuberculin-in order to avoid the danger of (more particularlyyglycerine poisoning.

; Special treatment-The active principle of tuberculin-for injectionat this stage is pro-- pared free from glycerine, preferably in the following manner: Five hundredcubic centi meters tuberculin is'heated to 80 centigrade,

then introducedinto a high cylindrical vessel of about fifteen-hundred c'ubiccentimeters capacity and seven hundred cubic centime-' ters ninetyper-cent. alcohol, cont-aining.0.8

percent. carbolic acid, added, constantly stirring. A light voluminous precipitate isformed almost immediately, which is induced to deposititself'by centrifuga'ting for-a quarter of an: hour. The supernatant liquid is then poured-0E from the. dark brown deposit and the latter'digested for twelve hours at 40 centigrade with sixty-per-cent. alcohol, con'-' taining 1-.5 per cent ammoniumsulphate in solution. Toinsure complete precipitation,

itisthen allowed to stand a further twelvehours atordinary temperature,whereupon the alcohol is carefully poured-"off and therprec-ipitate'brought upon a filter where it is washed-witha mixture consisting of; three parts ethyl alcohol. one part-benzene, one part chloroform,-,and 0.5 part amyl alcohol'and finally driedin an 'exsicca'tor Oven-Sulfuric After the first After acid. The preparation thu's obtained consists of a yellowish white powden'which dissolves fairly'easily in water. Seven milligrams of this preparation equal one cubic centimeter tuberculin in activity. As it commences to decompose after six to eight days when dissolved" in water, only the quantity required forimmediate use should be dissolved at one timer The animal already subjected to the preliminary treatment receives in aqueous so1u= tion fifteen milligrams of the preparation as a first in j ection and twenty-five milligrams as a second, which-is injected after forty-eight hours if no rise in temperature has meanwhile taken place. place, at least twenty-four hours mu'st be'al- "before further treatment is proceeded-witl-i. In the early stages of the treatment the animals exhibit great sensitivenessandreactab most regularly with slight feverafter'each injection. the dose gradually, only ten milligramsat a time; but after the animals have been accustomed to receive fifty milligrams ata single injection without-a marked general disturbfance of health-it is possible to progress more f quickly. From fifty milligrams the dose may be raised to seventy-five, the'nsuccessively to one hundred and one hundred and twenty 'five milligrams; but a pause of three-or four ,d'aysis necessary between" each injection. Naturally it is impossible to'draw hard and isesses-anindividuality of its own which refq'uiresindividual attention.

f The special treatment can be regarded as completed when for eachkilo body weight of f concentrated preparation,-fr'eefrom glycerine, {have been injected. r ifinal treatment:afteran'interval of about four :days. I

' Final traim'enh llor final treatnientdfem ;ployfa-preparation for injection which isrpref- Bouillon of" thecomposition above described is inoculated with tnbercle'bacilli under the iflpreca'utions defined above and allowed" to stand in the incubator at 40 centigradefor jtwo weeks- After this period has" elapsed auze, brought into, an agate mortar, and the whole well triturated'. Of this sterilize'd'liquid containing the dead 'remains'ofthe-"bacilli seven to ten cubic centin'ieters isinjected into each animal,- according to it"s-weight: In consequence0f this inoculation local sup'pura- @tionand abscesses are alwaysformed; but are quickly healed under antiseptic treatmen't. i

The animalshavi'ngi undergone the-above three treatments in-succ'ession are now kept, preferably,' about three or four weeks-before f'preparing the remedial serum; Then by If arise in temperature takes.

lowed to lapse after the temperature has fallen It is therefore necessary to-raise fast rules in this case, aseach' animal posthe animal two grams of the above-described" I now proceed to theerably produced in the following manner;

lthe bouillon is boiled'for twohours'overwire' drawing blood from them for the purpose of means of a trocar 0.5 to 0.75 liter of blood is drawn from the jugular vein under aseptic precautions, and after coagulation the serum is poured off, filtered through a Ohamberland filter, 0.4 per cent. carbolic acid added, and filled into sterilized bottles. It is then ready for therapeutical employment.

Experiments on animals have proved that this remedial serum prepared as above possesses specific antitoxic properties which cause it to heal light cases of tuberculosis and to stay the progress of severe and advanced cases of the disease.

The serum is characterized by its specific action upon tuberculous toxine, (tuberculin,) whereby it paralyz'es or neutralizes the action of tuberculin, and the Value of the serum as a therapeutical agent depends upon the degree of paralyzing or neutralizing actionit possesses.

The paralyzing or neutralizing action of the serum prepared in accordance with this invention may be demonstrated asfollows:

If into a guinea pig infected with tuberculosis thirty days previously an injection of a mixture of 0.5 gram tuberculin and three grams serum prepared in accordance with this invention is made, there is no appearance of any febrile reaction, and the animal will live for weeks after, whereas similar animals also infected with tuberculosis thirty days previously will die in a few hours from the injection of 0.5 gram tuberculin either alone or mixed with normal serum or diphtheria antitoxineserum.

What I claim, and desire to secure by Leta ters Patent, is-- 1. The process of manufacturing serum for the cure or retardation of the progress of tu= berculosis, which process consists in injecting into a living animal first tuberculin, secondly, tuberculin freed from glycerine, and thirdly a sterilized cultivation of tubercle bacilli containing the dead remains of the bacilli, in then, after an interval of several weeks, drawing blood from the animal, then coagulating it, and in then separating the serum from the blood, substantially as set forth.

2. The process of manufacturing serum'for the cure or retardation of the progress of tuberculosis, which process consists in injecting subcutaneously into a living animal and in approximately the quantities hereinbefore stated relatively to the Weight of the animal, firsttuberculin freed from glycerine, and then a sterilized cultivation of tubercle bacilli con taining the dead remains of the bacilli, in then after an interval of several weeks drawing blood from the animal and coagulating this blood, and in then separating the serum from the blood, substantially as set forth.

In witness whereof I have hereunto signed my name in the presence of two subscribing witnesses.

FRANZ NIEMANN.

. Witnesses:

RICHARD SCHMIDT, JOHN TOLLMAN. 

